EN 15786:2021
(Main)Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of Pediococcus spp. used as feed additive
Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of Pediococcus spp. used as feed additive
This document specifies general rules for the enumeration of pediococci in feeding stuffs (additives, premixtures and compound feeds excluding mineral feeds) that contain pediococci as a single microorganism component or in a mixture with other microorganisms. Applying the method to premixtures and compound feeds with critical amounts of copper demands a special procedure (see A.2). The document is not applicable to mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) 767/2009) [3]. There are different categories of feed samples:
a) Additives containing about 1010 colony forming units (CFU)/g;
b) Premixtures containing about 1011 CFU/kg;
c) Compound feeds, meal or pellets which contain about 109 CFU/kg.
Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von Pediococcus spp. als Futtermittelzusatzstoff
Dieses Dokument legt allgemeine Regeln für die Zählung von Pediokokken in Futtermitteln (Zusatzstoffe, Vormischungen und Mischfuttermittel mit Ausnahme von mineralischen Futtermitteln) fest, die Pediokokken als einzelnen mikrobiellen Bestandteil oder in einem Gemisch mit anderen Mikroorganismen enthalten. Die Anwendung des Verfahrens auf Vormischungen und Mischfuttermittel mit kritischen Kupfermengen erfordert ein besonderes Vorgehen (siehe A.2). Dieses Dokument ist nicht anwendbar auf mineralische Futtermittel, die als Ergänzungsfuttermittel definiert sind, die hauptsächlich aus Mineralstoffen zusammengesetzt sind und mindestens 40 % Rohasche enthalten (Verordnung (EG) Nr. 767/2009) [3]. Es gibt unterschiedliche Kategorien von Futtermittelproben:
a) Zusatzstoffe, die etwa 1010 koloniebildende Einheiten (KbE)/g enthalten;
b) Vormischungen, die etwa 1011 KbE/kg enthalten;
c) Mischfuttermittel, Mehle oder Pellets, die etwa 109 KbE/kg enthalten.
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et dénombrement des souches de Pediococcus spp. utilisées comme additifs pour l’alimentation animale
Le présent document spécifie des règles générales pour le dénombrement des pédiocoques présents dans les aliments pour animaux (additifs, prémélanges et aliments composés, à l’exception des aliments minéraux) qui contiennent des pédiocoques comme seul micro-organisme constitutif ou en mélange avec d’autres micro-organismes. L’application de la méthode aux prémélanges et aux aliments composés pour animaux présentant une teneur critique en cuivre nécessite de mettre en œuvre un mode opératoire spécial (voir A.2). Le présent document ne s’applique pas aux aliments minéraux, qui se définissent comme des aliments complémentaires constitués principalement de minéraux et contenant au moins 40 % de cendre brute (Règlement (CE) 767/2009) [3]. Il existe différentes catégories d’échantillons d’aliments pour animaux :
a) les additifs contenant environ 1010 UFC/g (UFC : unités formant colonie) ;
b) les prémélanges contenant environ 1011 UFC/kg ;
c) les aliments composés, farines ou granulés contenant environ 109 UFC/kg.
Krma: metode vzorčenja in analize - Določanje in štetje prisotnih Pediococcus spp., uporabljenih kot krmni dodatek
Ta mednarodni standard določa splošna pravila za štetje probiotičnih pediokokov v vzorcih krme (dodatki, premiksi in krma), ki vsebujejo pediokoke kot eno bakterijsko komponento ali v kombinaciji z drugimi mikroorganizmi. Ta standard se ne uporablja za mineralno krmo, ki je opredeljena kot dopolnilna krma, sestavljena predvsem iz mineralov, in vsebuje najmanj 40 % surovega pepela (Direktiva Sveta 79/373/EGS).
Obstajajo različne kategorije vzorcev krme:
a) dodatki, ki vsebujejo približno 10+10 enot, ki tvorijo kolonije (CFU)/g;
b) premiksi, ki vsebujejo približno 10+8 CFU/g;
c) krma, moka ali peleti, ki vsebujejo približno 10+6 CFU/g ter vključujejo popolno krmo in mlečne nadomestke.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2022
Nadomešča:
SIST EN 15786:2009
Krma: metode vzorčenja in analize - Določanje in štetje prisotnih Pediococcus
spp., uporabljenih kot krmni dodatek
Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of
Pediococcus spp. used as feed additive
Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von
Pediococcus spp. als Futtermittelzusatzstoff
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et
dénombrement des souches de Pediococcus spp. utilisées comme additifs pour
l’alimentation animale
Ta slovenski standard je istoveten z: EN 15786:2021
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 15786
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 15786:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Detection and enumeration of Pediococcus spp. used as
feed additive
Aliments des animaux: Méthodes d'échantillonnage et Futtermittel: Probenahme- und Untersuchungs-
d'analyse - Détection et dénombrement des souches de verfahren - Nachweis und Zählung von Pediococcus
Pediococcus spp. utilisées comme additifs pour spp. als Futtermittelzusatzstoff
l'alimentation animale
This European Standard was approved by CEN on 2 August 2021.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15786:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 6
5 Diluents and culture media . 7
5.1 Diluents . 7
5.2 Culture media . 8
6 Apparatus . 10
7 Sampling . 11
8 Preparation of test sample . 11
9 Procedure. 12
9.1 Samples with a single or multi-component added microflora . 12
9.2 Preparation of poured agar plates for spread plate method . 12
9.3 Preparation of MRS agar for pour plate method . 12
9.4 Preparation of the initial suspension and decimal dilutions . 12
9.5 Inoculation and incubation of plates . 13
9.6 Enumeration of colonies . 14
9.7 Confirmation . 14
10 Expression of results . 15
11 Precision . 16
11.1 General . 16
11.2 Interlaboratory study . 16
11.3 Repeatability . 16
11.4 Reproducibility . 16
12 Test report . 16
Annex A (informative) Notes on the procedure . 17
A.1 General . 17
A.2 Critical copper concentration . 17
Annex B (informative) Results of the interlaboratory study . 18
Bibliography . 20
European foreword
This document (EN 15786:2021) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2022, and conflicting national standards shall be
withdrawn at the latest by May 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 15786:2009.
The main changes compared to the previous edition are as follows:
— Amendment of the title;
— Extension of the scope of application to all Pediococci spp. used as feed additive;
— Updating of normative cross references; ®
— Supplement of phosphate buffered saline with Tween 80; ®
— Addition of the option to use Tween 80 supplemented phosphate buffered saline for the
preparation of the initial suspension as well as diluent for serial dilutions;
— Adjustment of the composition of the MRS agar to commercially available formulations;
-1
— Replacement of the required laboratory mixer with a rotation speed of 18 000 min to
-1
22 000 min by homogenization devices, for example according to EN ISO 7218, with a maximal
-1
requested rotation speed of 10 000 min ;
— Unification of the homogenization time for the preparation of initial suspensions to five minutes for
all feed matrices;
— Addition of the option to use a spiral plater for plating;
— Preparation of initial suspensions generally conducted with tempered tPBS;
— Addition of the pour plate method as an alternative cultivation technique;
— Addition of a procedure for the investigation of feeding stuffs containing high amounts of copper in
the informative Annex A;
— Adjustment of the range of accepted colony numbers for counting from '≥ 30 to ≤ 350' to '≥ 10
to ≤ 200' colonies per plate.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
Introduction
This methodology has been developed to enumerate pediococci used as feed additives to enable the
European Commission to control proper labelling of animal feeding products. It was compiled first during
the EU project SMT4-CT98-2235 “Methods for the official control of probiotics used as feed additives” [1].
The specified methodology was validated in an interlaboratory study [2]. The method is validated in this
project for one strain of Pediococcus acidilactici. It can be assumed that the method is suitable also for
other Pediococcus strains used as feed additives.
This method is not selective for pediococci used as feed additives, but can be applied to enumerate
Pediococci spp. in additives, premixtures and compound feeds assuming that the added pediococci are
present in far higher numbers than any other pediococci.
This method is not applicable for the detection of ubiquitous contaminants of Pediococcus spp. and any
other lactic acid bacteria in food and animal feeding stuffs.
1 Scope
This document specifies general rules for the enumeration of pediococci in feeding stuffs (additives,
premixtures and compound feeds excluding mineral feeds) that contain pediococci as a single
microorganism component or in a mixture with other microorganisms. Applying the method to
premixtures and compound feeds with critical amounts of copper demands a special procedure (see A.2).
The document is not applicable to mineral feeds which are defined as complementary feeding stuffs
composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) 767/2009) [3].
There are different categories of feed samples:
a) Additives containing about 10 colony forming units (CFU)/g;
b) Premixtures containing about 10 CFU/kg;
c) Compound feeds, meal or pellets which contain about 10 CFU/kg.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
Pediococci
Gram-positive, catalase negative, immobile cocci that grow under aerobic as well as under anaerobic
conditions
Note 1 to entry: This description is based on their characteristics as used for this document.
Note 2 to entry: Pediococci usually occur in pairs or tetrads, rarely in chains or singly, and divide along two planes
of symmetry. They form colonies fitting the description of these species on the specified selective media after
incubation of 48 h to 72 h at a temperature of 37 °C under aerobic or anaerobic conditions (see 9.7).
4 Principle
a) Preparation of sterile and dry poured agar plates or preparation of sterile liquid culture medium
tempered at 44 °C to 47 °C;
b) Drawing a representative test sample under aseptic conditions;
c) Preparation of the initial suspension with a tempered diluent to obtain a homogeneous distribution
of Pediococcus cells from the test portion;
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